Q Sepharose Fast Flow

i 71-7070-00

Edition AE

Q Sepharose Fast Flow

Q Sepharose? Fast Flow is a strong anion

exchanger with excellent flow properties and high

capacity for proteins of all pI values. The ion

exchange group is a quaternary amine which

remains charged and maintains consistently high

capacities over the entire working range, pH 2-12.

Q Sepharose Fast Flow belongs to the

BioProcess? Media family. BioProcess Media are

separation media developed, made and supported

for industrial scale - especially the manufacture

of healthcare products. With their high physical

and chemical stability, very high batch-to-batch

reproducibility, and Regulatory Support File

back-up, BioProcess Media are ideal for all stages

of an operation - from process development

through scale-up and into production.

Large quantities can be delivered at short notice.

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Table 1. Medium characteristics.

Type of ion exchanger:

Strong anion Total ionic capacity:

0.18-0.25 mmole/ml gel Available capacity*:Thyroglobulin (M r 669 000) 3 mg/ml HSA (M r 68 000)120 mg/ml α-lactalbumin (M r 14 300)110 mg/ml Bead structure:6% highly cross-linked agarose

Bead size range:45-165 μm

Mean particle size:90 μm

Linear flow rate:400-700 cm/h at 25 °C, 0.1 MPa (1 bar, 14 psi), XK 50/30

column, 15 cm bed height, mobile phase 0.1 M NaCl

Max. operating pressure:0.3 MPa (3 bar, 42 psi)

pH stability working range:2-12

long term:2-12

short term:1-14

Chemical stability:All commonly used aqueous buffers,1.0 M NaOH, 8 M urea,

8 M guanidine hydrochloride, 24% ethanol (tested at 40 °C

for 7 days).

Physical stability:Negligible volume variation due to changes in pH or ionic strength.

Autoclavable:

In 0.1 M NaCl at 121 °C for 30 min.*

The available capacity was de te rmine d in a 0.5 x 5 cm column at a line ar flow rate of 300 cm/h. Starting buffe r:0.05 M Tris, pH 8.3. Elution buffer: 0.05 M Tris, 2 M NaCl, pH 8.3.**The range s give n are e stimate s base d on our knowle dge and e xpe rie nce . Ple ase note the following:

pH stability, long te rm re fe rs to the pH inte rval whe re the ge l is stable ove r a long pe riod of time without adve rse e ffe cts on its subse que nt chromatographic pe rformance .

pH stability, short te rm re fe rs to the pH inte rval for re ge ne ration, cle aning-in-place and sanitization proce dure s.Preparation of the medium

Q Sepharose Fast Flow is supplied pre-swollen in 20% ethanol.Prepare a slurry by decanting the 20% ethanol solution and replace it with starting buffer in a ratio of 75% settled gel to 25% buffer.The starting buffer should not contain agents which significantly increase the viscosity. The column may be equilibrated with viscous buffers at reduced flow rates after packing is completed.

Packing Se pharose Fast Flow

1.Equilibrate all material to the temperature at which the

chromatography will be performed.

2.De-gas the gel slurry.

3.Eliminate air from the column dead spaces by flushing the

end pieces with buffer. Make sure no air has been trapped

under the column net. Close the column outlet with a few

centimeters of buffer remaining in the column.

4.Pour the slurry into the column in one continuous motion.

Pouring the slurry down a glass rod held against the wall of

the column will minimize the introduction of air bubbles.

5.Immediately fill the remainder of the column with buffer,

mount the column top piece onto the column and connect the column to a pump.

6.Open the bottom outlet of the column and set the pump to

run at the desired flow rate. Ideally, Fast Flow gels are

packed at a constant pressure not exceeding 3 bar (0.3 MPa) in XK columns. If the packing equipment does not include a pressure gauge, use a packing flow rate of at least 400 cm/h

(15 cm bed height, 25 °C, low viscosity buffer).

If the recommended pressure or flow rate cannot be obtained, use the maximum flow rate the pump can deliver. This should also give a reasonably well-packed gel.

Note:Do not exceed 75% of the packing flow rate in

subsequent chromatographic procedures.

7.Maintain the packing flow rate for 3 bed volumes after a

constant bed height is reached.

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Use of an adaptor

Adaptors should be fitted as follows:

1.After the medium has been packed as described above, close

the column outlet and remove the top piece from the column.

Carefully fill the rest of the column with buffer to form an

upward meniscus at the top.

2.Insert the adaptor at an angle into the column, ensuring that

no air is trapped under the net.

3.Make all tubing connections at this stage. There must be a

bubble-free liquid connection between the column and the

pump.

4.Slide the plunger slowly down the column so that the air

above the net and in the tubings is displaced by buffer.

Valves on the inlet side of the column should be turned in all directions during this procedure to ensure that air is removed.

5.Lock the adaptor in position, open the column outlet and

start the buffer flow. Pass buffer through the column at the

packing flow rate until the medium bed is stable. Re-position the adaptor on the medium surface as necessary. Equilibration

Before starting a run, make sure that the ion-exchange bed has reached equilibrium. This is done by pumping starting buffer through the column until the conductivity and/or pH of the effluent is the same as for that of the in-going solution.

The column is now equilibrated and ready for use.

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Binding

?The most common procedure is to let the molecules of interest bind to the ion exchanger and allow other molecules to pass through.

However, in some cases it may be more useful to bind

“contaminants” and let the molecules of interest flow through.?For adsorption, it is critical to choose a buffer with an appropriate pH. Please refer to Table 2. The ionic strength of the buffer should be kept low so as not to interfere with

sample binding. Recommended operating pH is within 0.5

pH units of the buffer’s pK

a and at least one pH unit above

the isoelectric point (pI) of the molecule of interest.

Table 2. Suggested buffers for use with Q Sepharose Fast Flow

Buffer Counter ion Concentration pKa (25°C) N-methylpiperazine Cl-20 mM 4.8 piperazine Cl-20 mM 5.7

HCOO-

L-histidine Cl-20 mM 6.2

bis-Tris Cl-20 mM 6.5

bis-Tris propane Cl-20 mM 6.8 triethanolamine Cl-20 mM7.8

CH

3COO-

Tris Cl-20 mM8.2

N-methyldiethanolamine SO

42-50 mM8.5

Cl-

CH

3COO-

diethanolamine Cl-

1,3-diaminopropane Cl-20 mM

ethanolamine Cl-20 mM

piperazine Cl-20 mM

1,3-diaminopropane Cl-20 mM

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Elution

Elution may be done using either an increasing salt gradient (linear or step) or an decreasing pH gradient (linear or step). Regeneration

Depending on the nature of the sample, regeneration is normally performed by washing with a high ionic strength buffer (e.g. 1-2 M NaCl) and/or decreasing pH, followed by re-equilibration in starting buffer.

In some applications, substances such as denaturated proteins or lipids do not elute in the regeneration procedure. These can be removed by cleaning in place procedures.

Cleaning in place (CIP)

?Remove ionically bound proteins by washing the column with

0.5 bed volumes of a 2 M NaCl solution, contact time 10-15

minutes, reversed flow direction.

?Remove precipitated proteins, hydrophobically bound proteins and lipoproteins by washing the column with 1 M NaOH

solution at a linear flow rate of approximately 40 cm/h,

contact time 1-2 hours, reversed flow direction. Wash with at least 3 bed volumes of starting buffer.

?Remove strongly hydrophobically bound proteins, lipoproteins and lipids by washing the column with 4 bed volumes of up to 70% ethanol or 30% isopropanol, reversed flow direction.

Apply increasing gradients to avoid air bubble formation

when using high concentrations of organic solvents.

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Alternatively, wash the column with 2 bed volumes of detergent in a basic or acidic solution. Use, for example, 0.1-0.5% non-ionic detergent in 0.1 M acetic acid. Wash at a low linear flow rate of approximately 40 cm/h, contact time 1-2 hours, reversed flow direction. After treatment with detergent always remove residual detergent by washing with 5 bed volumes of 70% ethanol.

In both cases wash with at least 3 bed volumes of starting buffer. Sanitization

Sanitization reduces microbial contamination of the medium bed to a minimum.

Wash the column with 0.5-1 M NaOH at a flow rate of approximately 40 cm/h, contact time 30-60 minutes, reversed flow direction.

Re-equilibrate the column with 3-5 bed volumes of sterile starting buffer.

Column performance is normally not significantly changed by the cleaning in place or sanitization procedures described above. Storage

It is recommended that the medium and packed column are stored for longer periods of time in 20% ethanol at 4 °C.

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